Molecular cloning and characterization of an inducible heat shock protein 70 gene in Chinese soft-shelled turtle (Pelodiscus sinensis)

摘要

Heat shock protein 70 (HSP70) is a class of highly conserved proteins which is involved in essential functions as molecular chaperones and in the acquired tolerance processes. The HSP70 family of proteins is encoded by two genes, a heat shock cognate 70 (Hsc70), and an inducible heat shock protein 70 (Hsp70) gene. In this work, the cDNA of Chinese softshelled turtle Pelodiscus sinensisHsp70 (designated PS-Hsp70)was cloned by the techniques of homological cloning and rapid amplification of cDNA end (RACE). The full length of PS-Hsp70 cDNAwas 2365 bp, consisting of a 5' -terminal untranslated region (UTR) of 64 bp, a 3' -terminalUTR of 393 bp, and an open reading frame (ORF) of 1908 bp encoding a polypeptide of 635 amino acids with a theoretical molecular weight of 69.81 kDa and an estimated isoelectric point of 5.37. BLAST analysis revealed that the PSHsp70 gene shared high similaritywith other Hsp70 genes. Three classical Hsp70 family signatures sequences were found in the PS-Hsp70 deduced amino acid sequence by InterPro analysis. The results indicated that the PS-Hsp70 was a member of the heat shock protein 70 family. A semiquantitiveRT- PCRmethodwas used to analyse the tissue specific expression of PS-Hsp70. The spatial mRNA expression of PS-Hsp70 gene were constitutively in the Pelodiscus sinensis tissues including muscle, liver, ovary, testis, kidney, spleen, lung and heart, with the highest level in testis.